Neuronal development in the Drosophila retina: The sextra gene defines an inhibitory component in the developmental pathway of R7 photoreceptor cells

Neuronal development in the Drosophila retina: The sextra gene defines an inhibitory component in the developmental pathway of R7 photoreceptor cells

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5271-5275, June 1992 Genetics Neuronal development in the Drosophila retina: The sextra gene defines an inhibitory component in the developmental pathway of R7 photoreceptor cells (eye development/sevenless) RONALD ROGGE, Ross CAGAN, ARINDAM MAJUMDAR, TOM DULANEY, AND UTPAL BANERJEE* Department of Biology and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90024 Communicated by Seymour Benzer, March 9, 1992 ABSTRACT Mutations in a gene called sextra (sxt) have been isolated. Loss of one copy of sxt promotes R7 photore- ceptor cell development in a genetically sensitized background, while loss of both copies results in precursors ofnon-neuronal cone cells transforming into R7 cells. The requirement for sxt function is cell-autonomous. The transformation of cone-cell precursors into R7 cells occurs independently of the sevenless signal. However, the R7 precursor becomes neuronal in an sxt/sxt mutant only in a wild-type sevenless background. The genetic analysis of sxt suggests that it plays an inhibitory role, preventing cone cells from becoming neuronal. Additionally, sxt functions in R7 precursors, but the sevenless sial is essential for specification of this fate, since oss of sextra alone is unable to impart a neural fate to this cell. Characterization ofa set ofgenes controlling R7 development has provided much insight into the process of induction and signal transduction (1-4). Genetic and molecular analysis of sevenless (sev) (5-8) and bride ofsevenless (boss) (9, 10) has demonstrated that R8 induces a neighboring cell to take on the R7 fate (9). In boss or sev mutants, R7 cells are missing within the eye. While the boss protein, a membrane-bound ligand, is required in the inducing R8 cell for normal R7 development (11), the sev gene encodes a tyrosine kinase receptor required in the R7 precursor for it to assume an R7 fate (12, 13). The boss protein has been shown to bind directly to the sevenless protein (11), initiating a molecular cascade that causes development of the R7 neuron.

Recently, two more genes, Son of sevenless (Sos) and Drasi, have been shown to participate in this signal- transduction pathway (14-16). Both gain- and loss-of- function mutations in Sos affect the development ofR7 cells. Sos functions downstream of sevenless and the Drosophila epidermal growth factorreceptorand encodes theDrosophila homolog of CDC25 of Saccharomyces cerevisiae. The CDC25 product has been shown to be an activator of Ras in yeast (17). It is likely that Sos functions as an activator of Drosophila Rasl.

A dominant mutation in Sos (called SosJc2) suppresses the phenotype ofa specific allele (sevE4) of sevenless (14). While sevE4 flies lack all R7 cells, in sevF-/sevE4;SosJc2/+ flies, R7 cells develop in a small fraction ofthe ommatidia. The sevE4 product is likely to have residual tyrosine kinase activity, and the SosJC2 product compensates for the partial loss of this kinase activity by hyperactivating the downstream molecule Ras (14, 16). This is a sensitized system where the fraction of ommatidia in which R7 develops is critically dependent on the dosage of other genes in the sevenless pathway. For example, in a sevE4/sevF4;SosJc2/+ mutant background, no R7 cells develop when one copy of boss or of Drasi is eliminated.

In this paper we characterize a gene called sextra (sxt), which also affects the development of R7 cells. However, unlike sev+, boss+, Sos+, and Drasl+, which promote the development of R7 cells, the wild-type function of sxt in the eye is to repress R7 fate. MATERIALS AND METHODS Genetic Analysis. The sxtBJ6] allele was isolated in a P-el- ement mutagenesis scheme essentially following Bier et al. (18), except that a P(ry+) element was used. For mosaic analysis, females carrying a P[w+] insertion at 78C/D were mated to either sxtlu6I/sxtBJ6' or sxtRI6I,bossl/ sxtMu6l,bossl males. Progeny from these crosses were irra- diated with -y-rays (1200 rads; 1 rad = 0.01 Gy) between 24 and 48 hr of development. Mosaic eyes were generated at a frequency of -0.01.

Histology. Preparation ofsamples fortransmission electron microscopy (TEM) was essentially as described (19), except that uranyl acetate staining en bloc was omitted. The sections were stained for 30 min with uranyl acetate and 10 min with lead citrate and were analyzed on a Phillips 300 electron microscope operating at 60 kV. For light-level microscopy, the fixation conditions were modified as described (9). To facilitate scoring of pigment granules, flies were exposed to bright light for 10 min before dissection. Cobalt sulfide staining was done as described (4).

Immunohistochemistry. Adult heads were dissected into halves and fixedfor 1 hrin 0.8%glutaraldehyde in phosphate- buffered saline. The retinas were transferred to fresh fixative, dissected out of the surrounding cuticle, allowed to fix for another 30 min, stained with antibodies (8), and embedded for sectioning (9). Eye imaginal discs were stained with mono- clonal antibody (mAb) 22C10 and processed for TEM (11). RESULTS Loss of One Copy of sxt Enhances SosJc2. R7 cells develop in 17% ofthe ommatidia in sevE4/sevE4;SosJc/+ flies (Table 1). This double mutant serves as a starting point for identi- fication of other genes involved in R7 development. Loss of one wild-type copy ofboss or Drasi reduces the suppression level to 0%. These genes have been shown to function as positive regulators of the pathway (11, 15, 16). Mutations in boss or Drasi are normally recessive; only in this sensitized system does a 2-fold reduction in their activity have an effect on R7 development.

Mutations in sxt enhance the sevE4/sevE4;SoSJC2/+ phe- notype. Loss ofa single copy ofsxt causes R7 cells to develop Abbreviation: mAb, monoclonal antibody. *To whom reprint requests should be addressed. 5271 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 89 (1992) Table 1. Levels of suppression Ommatidia % containing No. Genotype R7 cells scored sevF-/sevF-4;SosJC2/+ 17 1772 sevE4/sevF4;sxtBJ6I/+ 0 2259 sevF-/sevF-4;SosJC2/+ ;bossl/+ 0 2337 sevF-/sevF4;SosJC2/+ ;Df(Drasl)/+ 0 2175 sevF-/sevF4;SosJC2/+ ;sxtBJ61/+ 49 1934 Individual ommatidia were scored for the presence ofR7 by using the optical technique of pseudopupil (ref. 5 and references therein). The bossI mutation is a null allele. Since a Drasi point mutation was not available, Dft3R)by62, a deficiency including this locus, was used.

in 49%o ofthe ommatidia (Table 1), a substantial increase over the 17% level with two wild-type copies of sxt. In contrast, loss-of-function mutations in all other genes identified in this assay reduce the number of R7 cells that develop. This suggests an inhibitory role of sxt in the R7 developmental pathway. The ommatidia seen in sevE41sev-4;SosJc21+;sxt/+ flies never contain more than the one R7 cell, and this cell always occupies its wild-type position between R1 and R6. As shown later, the recessive phenotype ofsxt is to create additional R7 cells-hence the name sextra (for "seven extra"). Supernumerary R7 Cells Develop in sxt/sxt Eyes. In tan- gential sections of wild-type eyes, the rhabdomeres of outer photoreceptors R1-R6 are large and form a trapezoidal pattern in each ommatidium. These appear in both proximal and distal sections (Fig. 1 A and B) because they extend the entire length ofthe ommatidium. The rhabdomeres ofR7 and R8 project centrally and are smaller in size. The R7 rhab- domere is found distally (Fig. 1A), whereas R8 extends ,v 4; I - . t4' .

'V4 3. I( *.5 . + 1 W., proximally (Fig. 1B). The sxt mutation was made homozy- gous in order to assess its recessive phenotype. Sections of sxt/sxt eyes show that every ommatidium contains multiple centrally projecting rhabdomeres (Fig. 1C). Morphologi- cally, these are similar to R7 rhabdomeres in that they are small and project centrally and distally. Serial reconstruction of 61 ommatidia at the electron microscopic level showed that the development of R1-R6 is not affected in sxt. In 35 of the 61 ommatidia reconstructed, the rhabdomeres ofR1-R6 were found to be displaced, failing to extend the entire length of the ommatidium. However, in no case did an ommatidium contain fewer than six outer photoreceptor cells. In every ommatidium, a single rhab- domere was found to project proximally, in the position ofR8 (Fig. 1D). The number of R7-like cells in each ommatidium ranged from 2 to 6, although the majority contained either 3 or 4. The average number was 3.8.

To ascertain the identity of the extra cells, sxtBJ6' was crossed into a ninaE mutant background. The ninaE mutation causes the rhabdomeres of the outer photoreceptor cells (R1-R6) to degenerate, leaving the rhabdomeres ofR7 and R8 intact (Fig. 2A). In sxtBJ6I;ninaE double mutants, the rhab- domeres ofthe extra R cells do not degenerate (Fig. 2B). This implies that the extra cells have the identity ofeither R7 or R8. To distinguish between these two possibilities, the type of opsin expressed in the extra cells was determined. In Dro- sophila, two opsins, Rh3 and Rh4, are specific to R7 (20). A reporter gene controlled by the Rh4 promoter is expressed in a subset of R7 cells in wild-type eyes (Fig. 2C). In sxtBJ6I a similar fraction of the extra central cells express this R7- specific marker (Fig. 2D). This demonstrates that the central cells are of the R7 type.

A ~ ~ t 4%\!. W ,t s a A ~ ~ s-w-f B. ;4oi-* D ' P. J s; *qt. e ~ ~ S * *e S~ * 1. * :; .4..*,: > wbv..:s'.w,...D 's .' AIO,_ . t ~~~~e_.1 *t ) - - FIG. 1. Transmission electron micrographs of adult eyes. (A) Distal section through a wild-type ommatidium. Dark structures on the photoreceptor (R) cells are membrane specializations called rhabdomeres. Numbers 1-7 correspond to cells R1-R7. (B) Proximal section ofsame ommatidium as in A. The rhabdomere ofR8 is visible at this level. (C) Distal section through an sxtBJ6I/sxtBJ6I eye, showing multiple central photoreceptors. (D) Proximal section of same ommatidium as in C, showing a normal R8. (Bars = 2 ,um.) FIG. 2. Identity of the extra R cells in sxt/sxt. (Upper) Trans- mission electron micrographs. (A) Distal section through the eye of afly carryingtwo copies ofthe ninaEdeficiency Dft3L)I17e. The flies were aged for 5 days. Outer cell rhabdomeres have completely degenerated. The single rhabdomere visible in each ommatidium belongs to the R7 cell. (B) Distal section through the eye of an sxtBJ6I,DAf3L)117e/sxtBJ6l,Dft3L)I17e fly. The rhabdomeres of the extra cells do not degenerate in this background. (Lower) Light microscope sections of adult eyes. (C) Wild-type fly carrying the lacZ gene driven by the Rh4 promoter, stained with an anti-,- galactosidase antibody. A fraction of the R7 cells express the Rh4 promoter-driven reporter gene. (D) An sxtBJ611sxtm1 fly carrying the lacZ gene driven by the Rh4 promoter, stained with an anti- ,B-galactosidase antibody. A fraction of the R7 cells, including the extra central cells, stain positively with the antibody against the reporter gene product. (Bars = 5 Am.) 5272 Genetics: Rogge et A Airz -A .4 'II I .1.


Proc. Natl. Acad. Sci. USA 89 (1992) 5273 The extra R7 phenotype is strictly recessive in that sxt/+ flies have wild-type eyes. Due to the change in the internal morphology of the sxt/sxt flies, the external appearance of the eye is irregular or "rough." This external phenotype facilitated the mapping of the sxt locus. Genetic Mapping. The sxtBJ61 mutation was isolated in a P-element-induced mutagenesis and was mapped between the hairy and scarlet loci by standard genetic recombination techniques. Consistent with this mapping, a P element was detected by in situ hybridization on band 67C of the third chromosome (data not shown). The sxtsB6' mutation was mapped to the deficiency Dft3L)ACJ, which deletes bands 67A through 67D. The phenotype of sxtBJ6l/Dft3L)AC1 is identical to that of sxtBJ6I/sxtBJ6l at the light microscope level, in that the eyes are rough and extra R7 cells are seen, suggesting that sxtBJ6I is a null allele. Further, this same phenotype is seen when any one of 15 different imprecise excision alleles is placed over either sxtBJ6' or Df(3L)ACJ. Thirty-five precise excisions ofthe P element in sxt8161 were isolated. In each case, the sxt phenotype reverted to wild type, demonstrating that the P-element insertion was respon- sible for the sxt mutation.

Developmental Proffle. To view early events in ommatidial development, wild-type and sxt eye discs were stained with cobalt sulfide. In the wild-type disc, cobalt sulfide highlights the initial formation of six- or seven-cell preclusters at the morphogenetic furrow. This is followed by the loss ofone or two "mystery" cells (21), giving rise to a normal five-cell precluster consisting of R2, R3, R4, R5, and R8 (Fig. 3A). In sxt mutants, early development proceeds normally through the five-cell precluster stage (Fig. 3B). This is to be expected, since the fate of R8 and the outer cells is unaffected in this mutant.

To study later events, wild-type and sxt eye discs were stained with mAb 22C10, which recognizes a neural-specific antigen expressed by photoreceptor cells. The stained discs were sectioned tangentially and analyzed by electron micros- copy. In wild-type development, R1, R6, and finally R7 add to 5-cell preclusters, giving rise to mature 8-cell clusters. This is followed by the addition of4 non-neuronal cone cells. Cone cells in wild-type flies never stain with mAb 22C10 (Fig. 3 C and E). In sxt, R1, R6, and R7 add normally. However, cells in the position of cone-cell precursors begin expressing antigen 22C10, revealing their transformation to a neural fate (Fig. 3D). Thus, the sextra phenotype results from the transformation of cone-cell precursors into R7 cells. The cone-cell precursors are added to the developing sextra ommatidium in a strikingly ordered sequence (Fig. 3F; see also Fig. 5C). The anterior and posterior cone cells are the first to show transformation. This results in a 10-cell neural cluster in which cells are arranged in a stereotyped fashion. After a lag of several hours, a second pair of anterior and posterior cone-cell precursors are added, in addition to an equatorial and a polar cone-cell precursor. Some of these cells will express antigen 22C10 as well. This is in contrast to the transformation seen with ectopic boss expression (22), where development proceeds as in wild-type up to 28 hr, and only then do the cone-cell precursors assume R7 fates. Dependence on sev and boss. In sxt, as in wild type, the membrane-bound boss protein is restricted to R8 (data not shown). The cone-cell precursors that are converted into R7 in sxt do not contact R8 and therefore are not exposed to the ligand, yet they do become R7 cells. This implies that the development ofthe cone-cell precursors as R7 cells should be boss-independent. To test this genetically, double mutant combinations of sxt were made with boss as well as with sev. While flies mutant for sev or boss always lack R7 cells (Fig. 4B and C), the double mutants sev/sev;sxt/sxt and sxt,boss/ sxt,boss are similar in phenotype to sxt/sxt in that they display multiple R7 cells (Fig. 4 D-F).

A . B .Iei t .#o 41A *A D t PC * V0 4 R7 X . 4 PI R3 # - (s 9R4 F IP ~ ~ 8 H__ 4 g H G FIG. 3. Ommatidial assembly in sxt mutant eye discs. (A and B) Cobalt sulfide staining of third-instar larval eye discs. (Bars = 10 gm.) (A) Wild type. Cobalt sulfide stains apical membranes of differentiating cells. Staining commences as a dark band at the morphogeneticfurrow. Initial seven-cell clusters (arrow) resolve into five-cell preclusters of R8, R2, R5, R3, and R4 (arrowhead). (B) sxtBJ6]/sxtBJ6I. Pattern formation in sxt mutant discs proceeds normally through the stages described in A. (C-E) Transmission electron micrographs of eye disc stained with mAb 22C10. (Bars = 0.5 ,um.) (C) Developing wild-type ommatidium at the two-cone-cell stage. The anterior (AC) and posterior (PC) cone cells add to the cluster after the eight R cells have differentiated. Staining with mAb 22C10 can be seen in photoreceptor cells. The non-neuronal cone cells do not stain with this neural-specific antibody. (D) sxtBJ61/ sxtBJ6l ommatidium at a stage comparable to that shown in C. In this ommatidium, the cell in the position of the anterior cone cell (star) expresses the 22C10 neural antigen. (E and G) Wild-type ommatid- ium at the four-cone-cell stage. The membranes of the transmission electron micrograph in E have been traced in G for clarity. The cluster at this stage has 12 cells, R1-R8 and 4 cone cells. (F and H) sXtBJ61/sxt5J6' ommatidium at a stage comparable to that shown in Eand G. This clustercontains 14 cells: R1-R8, 3 cone-cell precursors converted into R7 cells (stars), and 3 other cells that do not stain with mAb 22C10 and are presumed to be cone cells.

Two different experiments were performed to determine whether the development ofall R7-like cells in sxt mutants is boss- and sev-independent. Adult eyes ofsevd2/sevd"2;sxt/sxt flies were sectioned, and 63 ommatidia were serially recon- structed at the EM level. The average number of R7 cells Genetics: Rogge et al. a:

Proc. Natl. Acad. Sci. USA 89 (1992) A** 't~i B Cute, * ~ ~ 4 I.' X ;it'.. A ~~~~~f nrik*p 0.X tj* P u; t' A" , I':. "'4~~ 4q * t d ai * ^ tw , o rmf1$*so 4 ~ Sk4,e * ', xS |i 4 . 4 .4 X. 1 IOWA ~ ' 4 F . 4 A, #'. a .14,v 'I A w 4 4 FIG. 4. Light microscope tangential sections through adult eyes. (A) Wild type. In this distal section, the rhabdomere projecting centrally in each ommatid- ium belongs to R7. (B and C) R7 cells are missing in sevd2/sev'2 (B) and boss'/boss' (C) mutant eyes. (D-F) Multiple R7 cells develop in sxtBJ61/sxtBJ61 (D), sevd2/sevd2;sxtBJ6)/sxtBJ6I (E), and sxtBJ6),bossl/ sxtBJ61,boss' (F) mutants. sev'12 and boss' are null alleles at the respective loci (6, 10). (Bars = 5 .tm.) developing per cluster was found to be 2.8. This number is different from the average of 3.8 seen in sxt/sxt flies, sug- gesting that 1 cell per ommatidium is sev-dependent. Fur- thermore, when sev/sev;sxt/sxt and boss,sxt/boss,sxt eye discs were stained using an elav antibody, which stains neuronal nuclei, no staining could be detected in the cell in the position of the R7 precursors at the light microscopic level. To determine whether the development of the R7 precursor into a neuron is sev-dependent in these double mutants, we scored developing sxt,boss/sxt,boss eye discs for the development of the ommatidium. The cell in the position of R7 did not stain with mAb 22C10 in a sxt,boss/ sxt,boss double mutant (Fig. 5). Ofthe 70 ommatidia that we scored at the EM level, one was found to show staining ofthe R7 precursor, while the other 69 precursors did not stain. Ommatidia were scored at a stage in which cone-cell precur- sors always express antigen 22C10 in a sxt/sxt mutant. We therefore conclude that the development ofthe R7 precursor is sev- and boss-dependent in a sxtBJ6' mutant background, whereas the cone-cell precursors are transformed into an R7 fate independently of sev.

The transformation of the cone cells results in functional R7 neurons since, like wild-type flies, sev/sev;sxt/sxt and sxt,boss/sxt,boss double mutants choose UV light over vis- ible light in a color choice paradigm (Table 2). sxt Function Is Cell-Autonomous. A mosaic patch of white cells of genotype sxt/sxt was generated in an otherwise red eye of genotype sxt/+. Along the boundary of the patch, mosaic ommatidia consisting of a mixture of red and white cells were screened. The aim was to determine which cells within an ommatidium must be white (i.e., sxt/sxt) for R7 cells to develop properly. This experiment was performed in two different ways.

First, mosaic patches were generated in a boss- back- ground to eliminate the inductive signal. Pigment granules in R7-containing mosaic ommatidia were scored. In 92 such ommatidia, all R7 cells were white (i.e., sxt/sxt). Thus the cells that take on R7 fate must be mutant for sxt. This implies that sxt function is autonomously required in precursor cells choosing between cone and R7 cell fates. The second mosaic experiment was done in a boss+ (i.e., wild-type) background. In this case, a cell in the position of the R7 precursor receives the boss- and sevenless-mediated signal, and therefore even when it is sxt+ (i.e., red), it would become R7. However, ifsxt is indeed autonomous, in no case should there be more than one red R7 cell in any ommatidium. This was indeed found to be the case. A total of 55 mosaic ommatidia containing multiple R7 cells were scored. Of these, 46 ommatidia had all white R7 cells. Each of the remaining 9 ommatidia contained a single red R7 cell, and no ommatidia were found with more than one red R7 cell. A Cl -w Ia type 7 ~ ~ 5 8 * ) I sextr..ra R82iig) R (a_ tt) ,_~t4 |R~a, c~cc In1 -S R2 -OR RS . .

FIG. 5. Development ofthe R7 precursor in an sxt,boss/sxt,boss double mutant. (A) mAb 22C10 staining of an ommatidium in this double mutant. (B) The cells shown in A have been traced for clarity. Staining can be seen in the cone-cell precursors (stars), while the R7 precursor (X) is clearly unstained. C, cone cells. (Bar = 1.0 um.) (C) Schematic representation of ommatidial assembly. Shading repre- sents mAb 22C10 staining. Unlike wild type, the anterior and posterior cone-cell precursors (ccp) stain in sxt and sxt,boss/ sxt,boss mutants. Four more cone-cell precursors are added later (stars), some of which transform into additional R7 cells. The R7 precursor always stains in sxt, but virtually never stains in sxt,boss/ sxt,boss (X). Hours refer to the time since initial cluster formation. D~~~ -.-.-A AL Ay t J 5274 Genetics: Rogge et al.

Proc. Natl. Acad. Sci. USA 89 (1992) 5275 Table 2. Color choice data No. to No. to Choice index, Genotype UV (A) visible (B) (A - B)/(A + B) Wild type 93 5 +0.90 sevF-/sevE4 6 105 -0.89 bossl/bossl 1 94 -0.98 sevF-4/sevF-4;sxtBJ61/sxtBJ61 3 +0.94 sxtBJ6',boss'/sxtBJ61,boss1 94 6 +0.88 Flies were allowed to choose between visible (550 nm) and UV (350 nm) light in a color choice test apparatus (6). Thirty flies were tested at a time; 20 sec was allowed for the test, with a gentle tapping at the end of 10 sec. Each group of flies was tested three consecutive times. No requirement for sxt was found in any of the other cell types in the ommatidium. In the two experiments described above, a total of 147 mutant mosaic ommatidia were serially reconstructed and were scored for the pigment phenotype of the outer cells and R8. In 64 of these ommatidia, the R8 cells were red (i.e., sxt+), and in the rest of the cases they were white. This implies that the genotype ofsxt in R8 is irrelevant for the development of R7 cells. Similarly, the outer cells in these ommatidia displayed no specific requirement forthe sxt genotype. The identity of an individual outer cell cannot be determined in the ommatidia that have extra R7 cells, since the normal trapezoidal pattern is often disrupted. However, in 13 of the ommatidia all six outer cells were red, implying that extra R7 cells can develop when all outer cells are wild-type for sxt. Finally, 4 ommatidia were found in which R1-R6 and R8 were red (i.e., sxt+) and all the R7 cells were white (i.e., sxt/sxt).

When taken together, the above results imply that wild- type sxt+ function is required only in the cells that have the potential to become R7, including the cone-cell precursors. DISCUSSION In a fly that is otherwise wild-type, the sxt mutation is recessive, i.e., a mutant phenotype is seen only in sxt/sxt flies. This phenotype results from the conversion ofcone-cell precursors into R7, leading to supernumerary R7 cells in each ommatidium. The effect of sxt on the development of R7, rather than cone-cell precursors, is more apparent in a sevE4/ sevE4;SosJc2/+ genetic background. In this sensitized sys- tem, the eye is neither completely wild type nor entirely lacking R7 cells, and loss of a single wild-type copy of sxt affects the number of R7 cells that develop. Thus, a signifi- cantly larger number of R7 cells are seen in sevE4/ sevF4;SosJC2I+;sxt/+ flies than in flies of the sevE4/ sevE4;SosJC2/+ genotype. However, in this case, no more than one R7 cell ever develops in a single ommatidium and cone-cell precursors are never converted to R7. A combination ofthese two results suggests that the normal function ofsxt is to inhibit a set ofcompetent precursor cells from assuming R7 fate. In wild type, this inhibition is presumably overridden in the R7 precursor by the boss- and sevenless-mediated signal. This model is consistent with the observations that nonspecific expression of boss (22) and overexpression ofan activated form of sevenless (23) lead to phenotypes that are similar to that seen in sxt/sxt flies. Presumably, in these cases, ectopic activation ofthe receptor can overcome the inhibitory effects of sxt in the cone-cell precursors.

Unexpectedly, the R7 precursor in a sextra mutant fails to become a neuron unless the sevenless signal is present, even though the cone-cell precursors transform into R7 indepen- dently of sevenless. Clearly, there is an early difference between the R7 and the cone-cell precursors. This difference may be due to the developmental timing of the cells or may resultfrom the signals they receive. The role ofsxt in the cone cells appears to be to inhibit their development as R7 cells. The failure of sxtkt6l to show complete epistasis to sev and boss, however, leaves the role of sxt in the development of the R7 cell unclear. One possible explanation ofthese results is that sxtBJ6I is not a null allele of the sxt locus. Given the consistent failure of the R7 precursor cell to develop as a neuron in an sxt,boss/sxt,boss double mutant, a more likely explanation is that sextra is not directly acted upon by the sevenless receptor.

Note Added in Proof. The mip mutation isolated independently by G. Buckles, Z. Smith, and F. Katz and the Gap] mutation isolated by U. Gaul, G. Mardon, and G. Rubin fail to complement sxt. This work was supported by a McKnight Scholars' award and an Alfred P. Sloan Foundation Fellowship to U.B. Research in U.B.'s laboratory is also supported by a grant (1 R01 EY08152-O1A1) from the National Institutes of Health. 1. Rubin, G. M. (1991) Trends Genet. 7, 372-377. 2. Banerjee, U. & Zipursky, S. L. (1990) Neuron 4, 177-187. 3. Ready, D. F., Hanson, T. E. & Benzer, S. (1976) Dev. Biol. 53, 217-240.

4. Tomlinson, A. & Ready, D. F. (1987) Dev. Biol. 120, 366-376. 5. Banerjee, U., Renfranz, P. J., Hinton, D. R., Rabin, B. A. & Benzer, S. (1987) Cell 51, 151-158. 6. Banerjee, U., Renfranz, P. J., Pollock, J. A. & Benzer, S. (1987) Cell 49, 281-291. 7. Hafen, E., Basler, K., Edstroem, J. E. & Rubin, G. M. (1987) Science 236, 55-63. 8. Tomlinson, A., Bowtell, D. D. L., Hafen, E. & Rubin, G. M. (1987) Cell 51, 143-150. 9. Reinke, R. & Zipursky, S. L. (1988) Cell 55, 321-330. 10. Hart, A. C., Kramer, H., Van Vactor, D. L., Paidhungat, M. & Zipursky, S. L. (1990) Genes Dev. 4, 1835-1847. 11. Kramer, H., Cagan, R. L. & Zipursky, S. L. (1991) Nature (London) 352, 207-212.

12. Bowtell, D. D. L., Simon, M. A. & Rubin, G. M. (1988) Genes Dev. 2, 620-634. 13. Basler, K. & Hafen, E. (1988) Cell 54, 299-311. 14. Rogge, R. D., Karlovich, C. A. & Banerjee, U. (1991) Cell 64, 39-48. 15. Simon, M. A., Bowtell, D. D. L., Dodson, G. S., Laverty, T. R. & Rubin, G. R. (1991) Cell 67, 701-716. 16. Bonfini, L., Karlovich, C. A., Dasgupta, C. & Banerjee, U. (1992) Science 255, 603-606. 17. Broek, D., Toda, T., Michaeli, T., Levin, L., Birchmeier, C., Zoller, M., Powers, S. & Wigler, M. (1987) Cell 48, 789-799. 18. Bier, E., Vaessin, H., Shepherd, S., Lee, K., McCall, K., Barbel, S., Ackerman, L., Carretto, R., Uemura, T., Grell, E., Jan, L. & Jan, Y. (1989) Genes Dev. 3, 1273-1287. 19. Van Vactor, D., Jr., Krantz, D., Reinke, R. & Zipursky, S. (1988) Cell 52, 281-290.

20. Montell, C., Jones, K., Zucker, C. & Rubin, G. (1987) J. Neurosci. 7, 1558-1566. 21. Tomlinson, A. & Ready, D. F. (1987) Dev. Biol. 123, 264-275. 22. Van Vactor, D. L., Cagan, R. L., Kramer, H. & Zipursky, S. L. (1991) Cell 67, 1145-1155. 23. Basler, K., Christen, B. & Hafen, E. (1991) Cell 64, 1069-1081. Genetics: Rogge et al.

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